Chronic Lymphocytic Leukemia with Stereotyped IGHV4-59/IGKV3-20 B Cell Receptors: Another Manifestation of Hepatitis C Virus-Associated B Cell Lymphoproliferation?.

Abstract Abstract 2331 Poster Board II-308 The hepatitis C virus (HCV) has been implicated in the development of B-cell lymphoproliferative disorders, including type II mixed cryoglobulinemia (MC-II) and B-cell lymphoma. MC-II is characterized by the presence of monoclonal IgM autoantibodies with rheumatoid factor (RF) activity. The monoclonal IgMs typically form immune complexes by binding polyclonal IgGs that exhibit anti-HCV reactivity. In a series of 6,196 patients affected by chronic lymphocytic leukemia (CLL), we have identified a subset of 12 cases sharing stereotyped mutated IGHV4-59/IGKV3-20 B cell receptors (BCRs) of the MD isotype (subset #13). Comparison of subset #13 heavy chain sequences to a comprehensive dataset of relevant public-database sequences revealed identical gene usage and remarkable junctional homology with the Ig sequence GenBank/U85234, the heavy chain of a RF detected in a healthy donor, as well as the sequence GenBank/AF303916, the clonotypic heavy chain from a CLL case with a history of HCV-associated MC-II. In addition, the light chain IGKV3-20/IGKJ1 stereotyped rearrangements in subset #13 were closely similar if not identical to the rearrangements expressed by clonally expanded IgM+κ+CD27+ B cells in HCV-associated MC-II. For both heavy and light chains, sequence similarities extended beyond junctional regions to shared, “stereotyped” somatic hypermutations across the entire IGHV and IGKV domain, respectively. We established viable and antibody-secreting heterohybridomas from the leukemic cells of a subset #13 case and confirmed the identity of the produced soluble antibody to the IG expressed by the CLL clone. ELISA tests against various antigens revealed that the soluble stereotyped IGHV4-59/IGKV3-20 antibody exhibited RF activity in vitro, while it was not reactive against HCV antigens. In conclusion, the present study for the first time provides evidence for the potential implication of HCV in the pathogenesis of at least a subset of CLL cases with distinctive stereotyped BCRs. The elucidation of the underlying immune mechanisms may pave the way for tailored anti-viral/anti-leukemic therapy for selected cohorts of patients that can be easily identified by molecular techniques during the diagnostic work-up. Disclosures: No relevant conflicts of interest to declare.

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Extensive Intraclonal Diversification in a Subgroup of Chronic Lymphocytic Leukemia Patients with Stereotyped IGHV4-34/IGKV2-30 B cell Receptors: Implications for Ongoing Interactions with Antigen.

Blood ◽

10.1182/blood.v114.22.2337.2337 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2337-2337

Author(s):

Lesley-Ann Sutton ◽

Efterpi Kostareli ◽

Anastasia Hadzidimitriou ◽

Nikos Darzentas ◽

Athanasios Tsaftaris ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Heavy Chain ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

B Cell Receptors ◽

Valuable Insight ◽

Cell Receptors ◽

Mutational Status ◽

The Impact

Abstract Abstract 2337 Poster Board II-314 Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen (Ag) recognition through the clonotypic B cell receptors (BCRs). However, it is still unclear whether Ag involvement is restricted to the malignant transformation phase or whether the putative Ag(s) may continuously trigger the CLL clone. Valuable insight into these issues may be gleaned from the study of intraclonal diversification (ID) within the immunoglobulin (IG) genes through ongoing somatic hypermutation (SHM). Definitive data regarding ID within IG genes in CLL remains limited and conflicting. In the present study we systematically explored the presence of ID within IG genes of CLL, not only at cohort level but also in subgroups defined by BCR stereotypy and IG gene mutational status. We thus conducted a large-scale subcloning study of both IG heavy and light variable genes, in a total of 1496 and 1008 subcloned sequences from 71 and 56 CLL cases, respectively. The analysis was intentionally biased to cases expressing IGHV4-34/IGKV2-30 IGs (subset #4) and IGHV3-21/IGLV3-21 IGs (subset #2) that exhibit distinctive, subset-biased SHM patterns. PCR reactions were run using the high-fidelity Accuprime Pfx polymerase and at least 14 colonies/case were analyzed. All “non-ubiquitous” sequence changes from the germline were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence from the same sample (ii) confirmed mutation (CM) - a mutation observed more than once among subcloned sequences from the same sample. Analysis of heavy chain sequences revealed that 40% (28/71) of cases carried intraclonally diversified IGHV-D-J genes with CMs amongst subclones, whilst 32% (23/71) of cases carried only UCMs. The remaining 28% (20/71) of cases carried sets of identical IGHV-D-J subcloned sequences. Although most cases showed no or low levels of ID, an intense and, likely, functionally driven ID was evident in selected cases, especially those belonging to subset #4. The distinct ID in subset #4 was statistically significant when compared to all other groups defined by IGHV gene usage and mutation status, BCR stereotypy or heavy chain isotype. Subsequent analysis of the clonotypic light chains revealed that the impact of ID was generally low, with the outstanding exception again relating to subset #4. In fact, of 22 IGKV-J rearrangements exhibiting CMs, 11 (50%) utilized the IGKV2-30 gene and notably 10/11 (91%) of these were expressed by subset #4 cases. In such cases, the expressed IGKV2-30 gene was affected by an active and precisely targeted ID, analogous to their partner IGHV4-34 gene. These findings suggest that the SHM mechanism may continuously operate in certain subsets of CLL patients, particularly those cases expressing stereotyped IGHV4-34/IGKV2-30 BCRs typical of subset #4. In such cases, the observed ID patterns attest to the very precise targeting of the SHM process and may be considered as evidence for a “stereotyped response” to an active, ongoing interaction with Ag(s). Disclosures: No relevant conflicts of interest to declare.

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IGHV1-69 B Cell Chronic Lymphocytic Leukemia Antibodies Cross-React with HIV-1 and Hepatitis C Virus Antigens as Well as Intestinal Commensal Bacteria

PLoS ONE ◽

10.1371/journal.pone.0090725 ◽

2014 ◽

Vol 9 (3) ◽

pp. e90725 ◽

Cited By ~ 23

Author(s):

Kwan-Ki Hwang ◽

Ashley M. Trama ◽

Daniel M. Kozink ◽

Xi Chen ◽

Kevin Wiehe ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Lymphocytic Leukemia ◽

Commensal Bacteria ◽

Virus Antigens ◽

Hiv 1 ◽

Cell Chronic Lymphocytic Leukemia

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Molecular evidence for EBV and CMV persistence in a subset of patients with chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors

Leukemia ◽

10.1038/leu.2008.379 ◽

2009 ◽

Vol 23 (5) ◽

pp. 919-924 ◽

Cited By ~ 47

Author(s):

E Kostareli ◽

A Hadzidimitriou ◽

N Stavroyianni ◽

N Darzentas ◽

A Athanasiadou ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Lymphocytic Leukemia ◽

B Cell Receptors ◽

Molecular Evidence ◽

Cell Receptors

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A novel chronic lymphocytic leukemia subset expressing mutated IGHV3-7-encoded rheumatoid factor B-cell receptors that are functionally proficient

Leukemia ◽

10.1038/leu.2012.238 ◽

2012 ◽

Vol 27 (3) ◽

pp. 738-740 ◽

Cited By ~ 27

Author(s):

R Hoogeboom ◽

T A Wormhoudt ◽

M R Schipperus ◽

A W Langerak ◽

D K Dunn-Walters ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Rheumatoid Factor ◽

Lymphocytic Leukemia ◽

B Cell Receptors ◽

Factor B ◽

Cell Receptors

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B-cell receptors expressed by lymphomas of hepatitis C virus (HCV)–infected patients rarely react with the viral proteins

Blood ◽

10.1182/blood-2013-10-532895 ◽

2014 ◽

Vol 123 (10) ◽

pp. 1512-1515 ◽

Cited By ~ 29

Author(s):

Patrick P. Ng ◽

Chiung-Chi Kuo ◽

Stanley Wang ◽

Shirit Einav ◽

Luca Arcaini ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

B Cell ◽

Viral Proteins ◽

B Cell Receptors ◽

B Cell Lymphomas ◽

Cell Receptors ◽

Key Points ◽

Experimental Approaches

Key Points We tested the hypothesis that B-cell lymphomas arising in HCV-infected patients express B-cell receptors specific to the virus. We analyzed the reactivity of these B-cell receptors with HCV proteins using several experimental approaches, none of which supported the hypothesis.

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Characteristics and outcome of B-cell chronic lymphocytic leukemia in hepatitis C virus-positive patients

Leukemia & Lymphoma ◽

10.1080/10428190600834412 ◽

2006 ◽

Vol 47 (11) ◽

pp. 2421-2423 ◽

Cited By ~ 1

Author(s):

Stefano Molica ◽

Rosanna Mirabelli ◽

Demetrio Misuraca

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Lymphocytic Leukemia ◽

Cell Chronic Lymphocytic Leukemia

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Subset-Specific Spectra of Recurrent Gene Mutations in Chronic Lymphocytic Leukemia with Stereotyped B-Cell Receptors

Blood ◽

10.1182/blood.v124.21.3320.3320 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3320-3320

Author(s):

Lesley-Ann Sutton ◽

Emma Young ◽

Panagiotis Baliakas ◽

Anastasia Hadzidimitriou ◽

Karla Plevova ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

B Cell ◽

High Frequency ◽

Gene Mutations ◽

Lymphocytic Leukemia ◽

B Cell Receptors ◽

Cell Receptors ◽

Recurrent Mutations ◽

Trisomy 12 ◽

Notch1 Mutations

Abstract Preliminary observations from essentially small patient series indicate that certain recurrent gene mutations may be enriched in subsets of chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptors (BcR). On these grounds, it could be argued that differential modes of immune signaling, in the context of subset-biased antigen-immunoglobulin (IG) interactions, may be associated with the acquisition and/or selection of certain genomic aberrations within various stereotyped CLL subsets. With this in mind, we here sought to explore the genetic background of 10 major stereotyped subsets which collectively account for ~11% of all CLL and represent both IGHV unmutated (U-CLL) and/or mutated (M-CLL) cases. We focused on recurrent mutations within the NOTCH1 (entire exon 34 or targeted analysis for del7544-45), TP53 (exons 4-9), SF3B1 (exons 14-16), BIRC3 (exons 6-9) and MYD88 (exon 5) genes. Overall, 647 cases were analyzed, belonging to the following major subsets: (i) U-CLL: #1 (the largest within U-CLL, clinically aggressive), n=139; #3, n=39; #5, n=22; #6, n=48; #7, n=74; #8, n=46; #59, n=19 and #99, n=18; (ii) M-CLL: #4 (the largest within M-CLL, particularly indolent), n=78; and, (iii) subset #2 (the largest overall, variable mutational status and clinically aggressive), n=164. All cases were devoid of MYD88 mutations, which was not surprising given that our cohort was predominantly composed of U-CLL. Mutations within the BIRC3 gene were either absent (#2, #4, #6 and #59) or rare (#1, #3, #5, #7, #8 and #99; frequency 1.5%-7%) with no clear bias to any subset. BIRC3-mutant cases frequently co-existed with either del(11q) or trisomy 12. NOTCH1 mutations were more frequent in subsets #1, #6, #8, #59 and #99 (frequency, 22%-32%), sharply contrasting subsets #2 or #3 (4% and 7%, respectively) (p<0.0001). Of note, although NOTCH1 mutations tended to coincide with trisomy 12 in certain subsets e.g. #1 and #8, their co-occurrence differed significantly with only 33% of NOTCH1mut subset #1 cases carrying trisomy 12 compared to 75% of NOTCH1mut subset #8 cases (p=0.036). Moving to SF3B1, we noted that subsets harboring NOTCH1 mutations were either absent for or carried few SF3B1 mutations, while the inverse was also true i.e. very high frequency of SF3B1 mutations in subsets #2 and #3, 45% and 36%, respectively. Almost 80% of mutations observed in subset #2 were localized to two codons (p.K700E: n=44/76, 58%: p.G742D: n=15/76, 20%) within the HEAT domain of the SF3B1 protein; p.K700E accounted for only 29% (4/14) of all SF3B1 mutations detected in subset #3 while p.G742D was absent (p=0.043 and p=0.068 respectively). Thus, although the functional relevance of these mutations is currently unknown, their high frequency and striking bias to subset #2 bodes strongly for their critical role in the pathobiology of subset #2. Finally, TP53 mutations were: (i) enriched in subsets #3 (11%) and #7 (19%) and, in contrast, absent or rare in subsets #5 (0%) and #6 (4%), despite all utilizing the IGHV1-69 gene (p=0.02); (ii) enriched in subset #1 (15%) and subset #99 (33%), a less populated subset that is highly similar to subset #1; and, (iii) very rare in subsets #2 and #8 (2% in both), the latter known to display the highest risk for Richter's transformation among all CLL. In conclusion, we confirm and significantly extend recent observations indicating that different CLL stereotyped subsets display distinct genetic makeup. These findings imply that distinctive modes of microenvironmental interactions, mediated by certain stereotyped BcRs, may be associated with selection or occurrence of particular genetic aberrations, with the combined effect determining both clonal and clinical evolution, and ultimately disease outcome. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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